Methods for delivering therapeutics across blood-barrier and for altering receptor binding affinity

ABSTRACT

The present invention relates to amphiphilic drug-oligomer conjugates capable of traversing the blood-brain barrier (“BBB”) and to methods of making and using such conjugates. An amphiphilic drug-oligomer conjugates comprise a therapeutic compound conjugated to an oligomer, wherein the oligomer comprises a lipophilic moiety coupled to a hydrophilic moiety. The conjugates of the invention further comprise therapeutic agents such as proteins, peptides, nucleosides, nucleotides, antiviral agents, antineoplastic agents, antibiotics, etc., and prodrugs, precursors, derivatives and intermediates thereof, chemically coupled to amphiphilic oligomers.

This application is a division of Ser. No. 09/134,803 filed Aug. 14,1998 now U.S. Pat. No. 6,703,381.

1. BACKGROUND OF THE INVENTION

1.1 Field of the Invention

The present invention relates to amphiphilic oligomer conjugates capableof traversing the blood-brain barrier (“BBB”) and to methods of makingand using such conjugates. The conjugates of the invention comprisetherapeutic agents such as proteins, peptides, nucleosides, nucleotides,antiviral agents, antineoplastic agents, antibiotics, etc., andprodrugs, precursors, derivatives and intermediates thereof, chemicallycoupled to amphiphilic oligomers.

1.2 Description of the Related Art

In the field of pharmaceutical and therapeutic invention and thetreatment of disease states and enhancement of physiological conditionsassociated with the CNS, a wide variety of therapeutic agents have beendeveloped, including proteins, peptides, nucleosides, nucleotides,antiviral agents, antineoplastic agents, antibiotics, etc., andprodrugs, precursors, derivatives and intermediates thereof.

Additionally, the many known neuroactive peptides offer additionalpossibilities for useful therapeutic agents. Such neuroactive peptidesplay important biochemical roles in the CNS, for example asneurotransmitters and/or neuromodulators. Delivery of this diverse arrayof peptides to the CNS provides many opportunities for therapeuticbenefit. For example, delivery of endogenous and synthetic opioidpeptides, such as the enkephalins, can be used to effect analgesia.

However, a number of obstacles currently limit the use of many compoundsfor use as CNS therapeutic agents.

First, the brain is equipped with a barrier system. The brain barriersystem has two major components: the choroid plexus and the blood-brainbarrier (BBB). The choroid plexus separates cerebrospinal fluid (CSF)from blood and the BBB separates brain ISF from blood.

The BBB has about 1000 times more surface area than the choroid plexusand is the primary obstacle to delivery of therapeutic compounds to theCNS. The BBB acts as a selective partition, regulating the exchange ofsubstances, including peptides, between the CNS and the peripheralcirculation. The primary structure of the BBB is the brain capillaryendothelial wall. The tight junctions of brain capillary endothelialcells prevent circulating compounds from reaching the brain ISF by theparacellular route. Furthermore, recent work suggests the existence of aphysiological barrier at the level of the basal lamina, in addition tothe barrier provided by the tight junctions. Kroll et al., Neurosurgery,Vol. 42, No. 5, p. 1083 (May 1998). Other unique characteristics of theBBB include lack of intracellular fenestrations and pinocytic vesiclesand a net negative charge on the luminal surface of the endothelium. Id.

The mechanisms by which substances may traverse the BBB may generally bedivided into active and passive transport mechanisms. Lipophilicmolecules readily traverse the BBB by passive transport or diffusionthrough the endothelial plasma membranes. In contrast, hydrophilicmolecules, such as peptides, typically require an active transportsystem to enable them to cross the BBB. Certain larger peptides, such asinsulin, have receptors on the luminal surface of the brain capillarieswhich act as active transcytosis systems.

Diffusion of many therapeutic compounds, such as peptides, across theBBB is also inhibited by size. For example, cyclosporin, which has amolecular weight of ˜1200 Daltons (Da), is transported through the BBBat a much lower rate than its lipid solubility would predict. Suchdivergence between lipid solubility and BBB permeation rates is probablydue to steric hinderances and is common where the molecular weight of acompound exceeds 800-1000 Da.

A further barrier to peptide delivery to the CNS is metabolicinstability. In particular, before peptides injected into the bloodreach the CNS, they must survive contact with enzyme degrading enzymesin the blood and in the brain capillary endothelium. BBB enzymes areknown to degrade most naturally occurring neuropeptides. Orallyadministered peptides face additional barriers discussed below.Metabolically stablized peptides may exhibit increased resistance tocertain enzymes; however, it has not been possible to protect peptidesfrom the wide range of peptide-degrading enzymes present in the bloodand BBB.

Another difficulty inherent in delivering peptides to the BBB is thatsuccessful transcytosis is a complex process which requires binding atthe lumenal or blood side of the brain capillary endothelium, movementthrough the endothelial cytoplasm, and exocytosis at the ablumenal orbrain side of the BBB. Peptides may bind to the lumenal membrane of thebrain capillary endothelium or undergo binding and endocytosis into theintracellular endothelial compartment without being transported into theCNS.

In any event, many currently existing drug substances, especiallypeptides, are unable to overcome these structural and metabolic barriersto enter the BBB in sufficient quantities to be efficacious. There istherefore a need for pharmaceutical compositions which can (1) withstanddegradative enzymes in the blood stream and in the BBB and (2) which canpenetrate through the BBB in sufficient amounts and at sufficient ratesto be efficacious.

Many attempts have been made in the art to deliver therapeuticcompounds, such as peptides, to the CNS with varying levels of success.Such attempts can generally be grouped into two categories: invasive andpharmacological.

Invasive delivery strategies include, for example, mechanicalprocedures, such as implantation of an intraventricular catheter,followed by pharmaceutical infusion into the ventricular compartment.Aside from general considerations relating to the invasiveness ofmechanical procedures, a major difficulty with mechanical approaches isthe lack of peptide distribution. For example, injection of peptidesinto the CSF compartment results in very little distribution beyond thesurface of the brain. This lack of distribution is due in part to rapidexportation of peptides to the peripheral circulation.

Another invasive strategy for delivering therapeutic compounds to theCNS is by intracartoid infusion of highly concentrated osmoticallyactive substances, such as mannitol or arabinose. Their high localconcentration causes shrinkages of the brain capillary endothelialcells, resulting in a transient opening of the tight junctions whichenable molecules to traverse the BBB. Such procedures have considerabletoxic effects, including inflammation, encephalitis, etc. Furthermore,such procedures are not selective: the opening of the tight junctions ofthe BBB permits many undesirable substances to cross the BBB along withthe therapeutically beneficial molecule. For a recent review of osmoticopening and other invasive means for traversing the BBB, see Kroll,Robert A. Neurosurgery, Vol. 42, No. 5, May 1998.

While the risks involved in these invasive procedures may be justifiedfor life-threatening conditions, they are generally not acceptable forless dramatic illnesses. There is therefore a need for less invasive,non-mechanical and safer means for enabling therapeutic compounds tocross the BBB.

As noted above, lipophilic substances can generally diffuse freelyacross the BBB. Accordingly, a common pharmacological strategy forenabling peptides to traverse the BBB is to chemically modify thepeptide of interest to make it lipid soluble. Hydrophilic drugsubstances have been derivatized with short chain or long chain fattyacids to form prodrugs with increased lipophilicity.

Prodrugs are biologically inert molecules which require one or moremetabolic steps to convert them into an active form. A difficulty withthe prodrug approach to crossing the BBB is that the cleavage necessaryto yield an active drug may not occur with sufficient efficiency andaccuracy to produce an efficacious amount of the drug.

There is therefore a need for modified stable therapeutic compounds,such as peptides, which are capable of traversing the BBB but whichretain all or part of their efficacy without requiring metabolic stepsto convert them into an active form.

A further difficulty with lipidized prodrugs is that they pass in andout of the CNS so readily that they may never reach sufficientconcentration in the CNS to achieve their intended function. Forexample, previous attempts have been made to engineer enkephalinconjugates which can traverse the BBB. See Partridge. W. M.,“Blood-Brain Barrier Transport and Peptide Delivery to the Brain,”Peptide-Based Drug Design: Controlling Transport and Metabolism, p. 277(1995). However, these strategies required the subcutaneous delivery offrequent and massive doses of peptide to induce analgesia. Frequentand/or massive dosing is inconvenient to the patient and may result inserious side effects.

There is therefore a need in the art for means for enabling therapeuticagents, such as peptides, to cross the BBB in a controlled manner whichpermits accumulation of sufficient quantities of the therapeutic in thebrain to induce the desired therapeutic effect.

Another pharmacological method for delivering peptides across the BBB isto covalently couple the peptide of interest to a peptide for which aspecific receptor-mediated transcytosis system exists. For example, itis theoretically possible to attach β-endorphin, which is not normallytransported through the BBB, to insulin to be transported across the BBBby insulin receptor-mediated transcytosis. Upon entry into the braininterstitial space, the active peptide (β-endorphin) is then releasedfrom the transport vector (insulin) to interact with its own receptor.

However, the difficulty with this system is designing a chimericmolecule which can become detached upon entry into the interstitialspace; to the inventor's knowledge, this has not yet been achieved.Additionally, the poor stoichiometry of the neuropeptide to the carriermolecule limits the mass of the target peptide. Furthermore,receptor-mediated cellular transport systems typically havephysiologically limited transport capacity. This is a rate-limitingfactor which can prevent entry of pharmaceutically active amounts ofpeptide.

There is therefore a need in the art for means for enabling therapeuticsubstances, such as peptides, to cross the BBB by diffusion so as toavoid the limitations inherent in receptor-mediated transport.

Other pharmacological strategies include using an active fragment of anative peptide; modification of a native peptide to increase blood-brainbarrier (BBB) transport activity; and delivery of a gene encoding theneuropeptide to the brain.

Oral administration is a desirable and convenient route ofadministration; however, orally delivered peptides must overcome aseries of barriers before they can enter the blook stream. Such peptidesmust survive proteolysis and the acidic environment of the stomach,gastric and pancreatic enzymes, exo- and endopeptidases in theintestinal brush border membrane.

There is therefore a need for orally administered peptides which canalso resist proteolytic enzymes in the blood and BBB and which cantraverse the BBB in sufficient quantities to provide broad distributionof drugs into the entire brain parenchyma.

Methionine-enkephalin and leucine-enkephalin are naturally occurringanalgesic pentapeptides. These peptides and their analogs are known toact as neurotransmitters or modulators in pain transmission. Theiranalgesic properties are short in duration. When administered byintracerebroventricular injection, the duration of their action is alsotransient.

These properties make the enkephalins attractive compounds for use astherapeutic agents, for mediating analgesia and providing a viablealternative to morphine. However, in order to deliver enkephalkinsacross the BBB, they must be protected against rapid degration byaminopeptidases and enkephalinases. Furthermore, since enkephalins arehydrophilic peptides, they must be modified to provide them withincreased lipophilic characteristics before they can passively diffuseacross the BBB into the CNS.

The attractive therapeutic properties of enkephalins have been known forsome time, and many investigators have attempted to enhance the abilityof enkephalins to traverse the BBB.

Schroder et al., Proc. Int. Symp. Control Rel. Biact. Material, Vol. 23,p. 611 (1996) teaches that Dalargin, a leu-enkephalin analogue can beincorporated in nanoparticles formed by polymerization ofbutylanoacrylate. The particles are coated with polysorbate, apenetration enhancer. Analgesic activity is obtained after intravenousadministration. Unlike the present invention, however, the Schroderpeptide must be chemically bound to the polymeric material or to thepolysorbate. The formulation is therefore a physical mixture of activedrug and polymeric material.

Tsuzuki et al., Biochem. Pharm. Vol. 41, p. R5 (1991) teaches thatanalogues of leu-enkephalin can be derivatized with adamantane moiety toobtain lipophilic enkephalin that shows an antinociceptive effect aftersubcutaneus administration. Modification at the N-terminus abolishesactivity while the derivative at the C-terminus through ester bondretains activity. It is postulated that the activity is obtained aftercleavage of the adamantane moiety. The derivative is therefore aprodrug, a concept not consistent with aspects of the present inventionin which the therapeutic conjugate retains the activity of the nativepeptide.

Prokai-Tatra, J. M. Chem, Vol. 39, p. 4777 (1996) teaches that aleucine-enkephalin analogue can be modified with chemical deliverysystem which is based on a retrometabolic drug design. The enkephaklinanalogue is derivatized with a dihydropyridine moiety at the N-terminusand a lipophilic moiety at the C-terminus. After intravenousadministration of the conjugate, analgesic response is observed. It ispostulated that the lipophilic modification at the C-terminus enablespenetration into the CNS, while the dihydropyridine moiety undergoesoxidative transformation to generate a charged moiety which restrictsthe peptides from effluxing into the circulatory system. Cleavage of thepeptide from this moiety restores the observed analgesic activity. Thederivatzed peptide is inactive and regains activity only after metabolictransformation. The product is therefore a pure prodrug, requiringmetabolic transformation to transform it into an active form.

U.S. Pat. No. 4,933,324 to Shashoua teaches that certain natural fattyacids can be conjugated to neuroactive drugs. A highly unsaturated fattyacid of twenty-two (22) carbon chain length is particularly preferred.Administration of the conjugate shows absorption into the brain. As isthe case with adamantane conjugation, this approach requires metabolictransformation of the prodrug conjugate of enkephalin to restore theactivity of the enkephalin peptide.

There is therefore a compelling need in the art for pharmaceuticallyacceptable and effective therapeutic/diagnostic compositions capable oftraversing the BBB without substantial loss or diminution of theirtherapeutic or diagnostic character.

2. SUMMARY OF THE INVENTION

The present invention broadly relates to therapeutic and/or diagnosticdrug-oligomer conjugates wherein a drug molecule is covalently bonded toan oligomer to form an amphiphilic conjugate. In one aspect, theoligomer comprises at least one lipophilic moiety and at least onehydrophilic moiety, and the size and nature of the amphiphilic andlipophilic moieties is so selected as to impart an amphiphilic nature tothe resulting conjugate.

The present invention relates generally to amphiphilic drug-oligomerconjugates capable of traversing the BBB and to methods of making andusing such conjugates.

In one aspect, the therapeutics are neuroactive drugs, proteins,peptides and especially enkephalin analogues. The conjugates are stablein the environment of the bloodstream and resist degradation by theenzymes of the BBB and in the CNS. Furthermore, the conjugates readilytraverse the BBB.

In one aspect, the drug-oligomer conjugates produce their intendedpharmacological effect without undergoing metabolic cleavage of theoligomer.

In another aspect, the lipophile and hydrophile are connected by alabile, hydrolyzable bond. When the bond is hydrolyzed in the CNS, thehydrophile remains attached to the drug.

The amphiphilic oligomers are composed of lipophilic and hydrophilicmoieties. The lipophilic moieties are preferably natural fatty aids oralkyl chains. Preferably, the fatty-acid moiety is a straight chainmolecule having (saturated or unsaturated) carbon atoms and suitablyranges from four (4) to twenty-six (26) carbon atoms. Most preferably,the fatty acid has from fourteen (14) to twenty-two (22) carbon atoms.

The hydrophilic moieties are preferably small segment of polyethyleneglycol (PEG), preferably having 1-7 PEG units, and more preferably 1-5PEG units. The length and composition of the lipophilic moieties and thehydrophilic moieties may be adjusted to obtain desired amphiphilicity.

In another aspect, a cholesterol or adamantane moiety is substituted forstraight chain fatty add portion of the oligomers.

Examples of preferred oligomers are as follows:

-   -   wherein n=3 to 25 and m=1 to 7;    -   wherein n=3 to 25 and m=1 to 6;    -   wherein n=3 to 25, m=1 to 7 and X=O;    -   wherein m=0 to 5 and R=cholesterol or adamantane; or    -   wherein m=0 to 5;    -   wherein m=0 to 7;    -   wherein m=1 to 7 and X=N or O.

Other unsaturated fatty acid moieties which can be used according to thepresent invention include oleic, linoleic, and linolenic.

For example, in one aspect the lipophile and hydrophile are connected byhydrolyzable bonds. It is preferred provide hydrolyzable bonds betweenthe fatty acid and hydrophilic moieties. This permits hydrolysis tooccur after penetration into the CNS, thus releasing the active peptideswith the hydrophilic group still attached to the peptide. As a result,the peptide acquires a more hydrophilic character and efflux to thecirculatory system is thereby hindered.

Exemplary conjugates having non-hydrolyzable bonds are as follows:

-   -   DHA MET-ENKEPHALIN-LYS SEQ ID NO: 1    -   LINOLEIC MET-ENKEPHALIN-LYS SEQ ID NO: 1    -   CETYL MET-ENKEPHALIN-LYS SEQ ID NO: 1

In another aspect, the lipophile and hydrophile are connected byhydrolizable bonds. For example:

-   -   CHOLESTEROL MET-ENKEPHALIN-LYS SEQ ID NO: 1    -   PALMITATE-TEG MET-ENKEPHALIN-LYS SEQ ID NO: 1    -   DI-PALMITATE-TEG MET-ENKEPHALIN-LYS SEQ ID NO: 2

In one aspect, the covalent bond between the oligomer and the drug ispreferably amide (a carboxy group of the oligomer is linked to an aminegroup of the peptide drug), or carbamate (an chloroformate group of theoligomer is linked to an amine group of the peptide drug). In general,the derivitizable amine group of the peptide is the amine of theN-terminus or a nucleophilic amino residue, usually found on the epsilonamino residue of a lysine residue.

In another aspect, an ester (a carboxy group of the peptide iscovalently coupled to a hydroxyl group of the oligomer or a carboxygroup of the oligomer is covalently coupled to a hydroxyl group of thedrug), amide (a carboxy group of the oligomer is linked to an aminegroup of the drug) or carbamate (an chloroformate group of the oligomeris linked to an amine group of the drug) bond is provided fornon-peptide drugs.

For the enkephalin analoges the preferred peptides are leu-enkephalinlysine SEQ ID NO: 50 and met-enkephalin lysine SEQ ID NO: 49. The aminoside chain of the lysine is preferably utilized in bonding.

The amphiphilic drug-oligomer conjugate may comprise multiple oligomersof differing compositions.

In another aspect, the amphiphilic drug-oligomer conjugates moieties areconfigured as follows:

-   -   R—OCH₂CH₂OCH₂C(O)OCH₂CH₂CH₂OCH₂CH₂CH₂NH-Enkephalin SEQ ID NO: 1;        or    -   R—OCH₂CH₂OCH₂C(O)OCH₂CH₂NH-Enkephalin SEQ ID NO: 1.

Wherein R=alkyl₁₋₂₆, cholesterol or amantane.

In another aspect of the amphiphilic oligomer moieties are sugarmoieties coupled to natural fatty acids and segments of polyethyleneglycol. The PEG moiety serves to increase the amphiphilicity of thefatty sugar. Examples of arrangements including sugar moieties areprovided in FIGS. 1A-1C.

In another aspect, PEG is used as a spacer group in the amphiphilicconjugate and the length and number of the PEG moieties can be varied torefine the amphiphilicity of the conjugate. Increasing the number ofPEGs increases the hydrophilicity of the conjugate.

In another aspect of the invention, a proline or alanine is added to theN-terminus of the peptide. In a preferred aspect, a praline or alanineis added to the N-terminus of an enkephalin peptide and the oligomermoiety is coupled to the N-terminus of the proline or alanine residue.

After absorption into the central nervous system, the esters of thefatty sugar are hydrolysized leaving a hydrophilic moiety. Efflux ishindered and the brain aminopeptidases cleave the proline or alanineportion leaving the peptide to regain full activity.

The invention also provides a pharmaceutical composition comprising anamphiphilic drug-oligomer conjugate and a pharmaceutically acceptablecarrier.

In another aspect, a pharmaceutical composition is provided comprising(1) a mixture of an enkephalin conjugate according to the presentinvention wherein the enkephalin peptide has proline or alanine added toits N-terminus and an enkephalin conjugate according to the presentinvention which does not have a proline or alanine added to theN-terminus, and (2) a pharmaceutical carrier. This aspect provides afaster acting sustained dose of enkephalin.

The invention also provides methods of administering a conjugate of theinvention.

The invention further provides assays, both in vitro and in vivo, fortesting the efficacy of the conjugates of the invention.

Other objects and further scope of applicability of the presentinvention will become apparent from the detailed description givenhereafter. It should be understood that the detailed description andspecific examples, while indicating preferred embodiments of theinvention, are given by way of illustration only, since various changesand modifications will become apparent to those skilled in the art fromthe detailed description.

2.1 Definitions

As used herein, the term “lipophilic” means the ability to dissolve inlipids and/or the ability to penetrate, interact with and/or traversebiological membranes.

As used herein, the term, “lipophilic moiety” or “lipophile” means amoiety which is lipophilic and/or which, when attached to anotherchemical entity, increases the lipophilicity of such chemical entity,e.g., fatty acid, cholesterol.

As used herein, the term “hydrophilic” means the ability to dissolve inwater.

As used herein, the term “hydrophilic moiety” or “hydrophile” refers toa moiety which is hydrophilic and/or which when attached to anotherchemical entity, increases the hydrophilicity of such chemical entity,e.g., sugars, PEG.

As used herein, the term “amphiphilic” means the ability to dissolve inboth water and lipids.

As used herein, the term “amphiphilic moiety” means a moiety which isamphiphilic and/or which when attached to a peptide or non-peptide drugincreases the amphiphilicity of the resulting conjugate, e.g., PEG-fattyacid oligomer, sugar-fatty acid oligomer.

As used herewith, the term “neuroactive drug” is used broadly toencompass any peptide or other drug having an activity within the CNS,e.g., enkephalin, enkephalin analogues.

As used herein the term “peptide” is intended to be broadly construed asinclusive of polypeptides per se having molecular weights of up to about10,000, as well as proteins having molecular weights of greater thanabout 10,000.

As used herein, the term “covalently coupled” means that the specifiedmoieties are either directly covalently bonded to one another, or elseare indirectly covalently joined to one another through an interveningmoiety or moieties, such as a bridge, spacer, or linkage moiety ormoieties.

As used herein, the term “drug” means a substance intended for use inthe diagnosis, characterization, cure, mitigation, treatment, preventionor allaying the onset of a disease, disease state, or otherphysiological condition or to enhance normal physiological functioningin humans and/or in non-human animals.

3. BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C: Formulae 8-10; amphiphilic oligomers of the presentinvention where in the lipophile is a sugar. In 1 b and 1 c, PEG is usedas a spacer group. In 1A-1C a proline residue is added at the N-terminusof the enkephalin peptide. Hydrolysis cleaves the lipophilic moietiesand generates a hydrophilic sugar-drug conjugate. Brain peptidasescleave the sugar moiety to generate a free peptide.

FIG. 2: Compares the stability of the cetyl-PEG₂-enkephalin-lys (SEQ IDNO:1) conjugate (non-hydrolyzable) to unconjugated enkephalin (SEQ IDNO:48) in rat brain homogenate. (2% Rate Brain in PBS buffer, pH 7.4,37° C.; Peptide=60 μg/mL).

FIG. 3: Compares the stability of the cetyl-PEG₃-enkephalin (SEQ IDNO:1) conjugate (non-hydrolyzable) to unconjugated enkephalin (SEQ IDNO:47) in rat brain homogenate. (2% w/v Rat brain in PBS buffer, pH 7.4,37° C.; Peptide=60 μg/mL).

FIG. 4: Compares palmitate-PEG₃-Enk (SEQ ID NO:1) conjugate(hydrolyzable) to unconjugated enkephalin (SEQ ID NO:47) in rat brainhomogenate. (2% w/v Rat Brain in PBS buffer, pH 7.4, 37° C.; Peptide=60μg/mL).

FIGS. 5 a-5 d: HPLC data showing extraction of conjugate fromhomogenized rat brain. HPLC conditions: Column: C-18: Solvent: solventA=IPA; solvent B=Water+0.1% TFA; Gradient: linear.

FIG. 6: Graph demonstrating competitive binding betweencetyl-PEG₂-enkephalin (SEQ ID NO:1) conjugate and naloxone, an Opioid μreceptor agonist.

FIG. 7: Graphic comparison of analgesic effect of cetyl-PEG₂-enkephalin(SEQ ID NO:1) with clonidine (a morphine substitute).

FIG. 8: Table showing results of receptor binding assays for variousconjugates according to the present invention. Data are based on percentinhibition at a concentration of 100 nM. The radioligand was DAMGO([D-Ala2,N-Me-Phe-Gly5-ol]enkephalin) and naloxone served as thereference.

FIG. 9: Exemplary synthetic scheme for an oligomer according to thepresent invention.

FIG. 10: Exemplary synthetic scheme showing attachment of an oligomer toan enkephalin peptide according to the present invention.

4. DETAILED DESCRIPTION OF THE INVENTION

For clarity of disclosure, and not by way of limitation, the detaileddescription of the invention is divided into the subsections whichfollow.

The present invention relates generally to amphiphilic drug-oligomerconjugates capable of traversing the BBB and to methods of making andusing such conjugates.

The drugs are preferably neuro-active drugs, proteins, peptides andespecially enkephalin analogues. The conjugates are stable in theenvironment of the bloodstream and resist degradation by the BBB. Theconjugates readily traverse the BBB.

In one aspect, the conjugates produce their intended pharmacologicaleffect without requiring metabolic cleavage of the oligomer. Whencleavage of the oligomer occurs, the drug retains activity.

The amphiphilc oligomers are composed of lipophilic and hydrophilicmoieties. The hydrophilic moieties are preferably natural fatty aids oralkyl chains. The lipophilic moieties are preferably small segments ofPEG, having 1 to 7 PEG moieties, and preferably having 1 to 5 PEGmoieties. The length and composition of the lipophilic moieties and thehydrophilic moieties may be adjusted to obtain desired amphiphilicity.For example, the carbon chains of the fatty acid or alkyl moieties maybe lengthened to increase lipophilicity, while PEG moieties may belengthened to increase hydrophilicity.

Preferably, the fatty-acid moiety is a straight chain molecule havingsaturated and unsaturated carbons and ranges from four (4) to twenty-six(26) carbon atoms. Most preferably, the fatty acid has from fourteen(14) to twenty-two (22) carbon atoms.

A cholesterol or adamantane moiety can be substituted for straight chainfatty acid as the lipophilic portion of the oligomers.

Examples of preferred oligomers are as follows:CH₃(CH₂)_(n)(OC₂H₄)_(m)OH  (Formula 1);

-   -   wherein n=3 to 25 and m=1 to 7;         CH₃(CH₂)_(n)(OC₂H₄)_(m)OCH₂C₂H  (Formula 2);    -   wherein n=3 to 25 and m=1 to 6;        CH₃(CH₂)_(n)CO(OC₂H₄)_(m)OH  (Formula 3);    -   wherein n=3 to 25, and m=1 to 7;        R—(OC₂H₄)_(m)CH₂CO₂H  (Formula 4)    -   wherein m=0 to 5 and R=cholesterol or adamantane; or        R—OCO(C₂H₄O)_(m)CH₂CO₂H  (Formula 5);    -   wherein m=0 to 5;        CH₃(CH₂CH═CH)₆(CH₂)₂CH₂(OC₂H₄)_(m)OH  (Formula 6);    -   wherein m=1 to 7;        CH₃(CH H═CH)₆(CH₂)₂CO(OC₂H₄)_(m)OH  (Formula 7);    -   wherein m=1 to 7.

Other unsaturated fatty acid moieties which can be used according to thepresent invention include oleic, linoleic and linolenic.

In certain instances, it is preferred to provide hydrolyzable bondsbetween the polyethylene glycol and the fatty acid moieties. Thispermits hydrolysis to occur after penetration into the central nervoussystem, thus releasing the active peptides with the polyethylene glycolgroup still attached to the peptide. The peptides acquire a morehydrophilic character and efflux to circulatory system is therebyhindered.

The covalent bond between the oligomer and the drug is preferably amide(a carboxy group of the oligomer is linked to an amine group of thepeptide), or carbamate (a chloroformate group of the oligomer is linkedto an amine group of the peptide).

For non-peptide drug, the bond is preferably ester (a carboxy group ofthe peptide is covalently coupled to a hydroxyl group of the oligomer ora carboxy group of the oligomer is covalently coupled to a hydroxylgroup of the drug), amide (a carboxy group of the oligomer is linked toan amine group of the drug) or carbamate (a chloroformate group of theoligomer is linked to an amine group of the drug). For the enkephalinanalogues, the preferred peptides are leu-enkephalin lysine andmet-enkephalin lysine. The amino residue of the lysine is preferablyutilized in bonding.

Other preferred amphiphilic moieties are sugar moieties, coupled tonatural fatty acids and segments of polyethylene glycol. The PEG moietyserves to increase the amphiphilicity of the fatty sugar.

The length and number of the PEG moieties can be varied to refine theamphiphilicity of the conjugate. Increasing the number of PEGs increasesthe hydrophilicity of the resulting oligomer.

In certain instances, it is preferred to modify the N-terminus of anenkephalin with proline or alanine before attaching the oligomer. Afterabsorption into the central nervous system, the esters of the fattysugar are hydrolysized leaving hydrophilic moiety. Easy efflux ishindered and the brain aminopeptidases cleave the proline or alanineportion leaving the peptide to regain full activity.

Where the hydrophilic moiety is a sugar, it is preferred that the sugaris a monosaccharide. The sugar may be an amino sugar or a non-aminosugar.

In another aspect, the oligomer is attached to the C-terminus of thepeptide drug. For example:

-   -   R—OCH₂CH₂OCH₂C(O)OCH₂CH₂CH₂OCH₂CH₂CH₂NH-Enkephalin SEQ ID NO: 1;        or    -   R—OCH₂CH₂OCH₂C(O)OCH₂CH₂NH-Enkephalin SEQ ID NO: 1.

Wherein R=alkyl₁₋₂₆, cholesterol or amantane.

In another aspect, the oligomer is attached at the N-terminus of thepeptide drug. For example:

It will be appreciated by one of skill in the art that the oligomers maybe attached at the carboxy terminus or at a constituent of an amino acidside chain, such as at the amino group of lysine.

The present invention broadly relates to therapeutic and/or diagnosticconjugates wherein the therapeutic and/or diagnostic molecule iscovalently bonded to an oligomer to form an amphiphilic conjugate. Inone aspect, the oligomer comprises at least one lipophilic moiety and atleast one hydrophilic moiety, and the size and nature of the twomoieties is so selected as to impart an amphiphilic nature to theresulting conjugate.

Exemplary oligomers according to the present invention are as follows:CH₃(CH₂CH═CH)₆(CH₂)₂CH₂(OC₂H₄)_(m)OH,

-   -   where m=1 to 7;        CH₃(CH₂CH═CH)₆(CH₂)₂CO(OC₂H₄)_(m)OH,    -   where m=1 to 7;        CH₃(CH₂CH═CH)₆(CH₂)₂CONHCH₂CH₂(OC₂H₄)_(m)OH,    -   where m=1 to 6;         CH₃(CH₂CH═CH)₆(CH₂)₃(OC₂H₄)_(m)OCH₂COOH,    -   where m=1 to 6;        CH₃(CH₂CH═CH)₆(CH₂)₂CO(OC₂H₄)_(m)OCH₂COOH,    -   where m=1 to 6;        CH₃(CH₂)₇CH═CH(CH₂)₈(OC₂H₄)_(m)OH,    -   where m=1 to 7;        CH₃(CH₂)₇CH═CH)(CH₂)₇CO(OC₂H₄)_(m)OH,    -   where m=1 to 7;        CH₃(CH₂)₇CH═CH(CH₂)₇CONHCH₂CH₂(OC₂H₄)_(m)OH,    -   where m=1 to 6;        CH₃(CH₂)₇CH═CH(CH₂)₈(OC₂H₄)_(m)OCH₂COOH,    -   where m=1 to 6;        CH₃(CH₂)₇CH═CH(CH₂)₇CO(OC₂H₄)_(m)OCH₂CH₂OH,    -   where m=1 to 6;        CH₃(CH₂)₄CH═CHCH₂CH═CH(CH₂)₇CH₂(OC₂H₄)_(m)OH,    -   where m=1 to 6;        CH₃(CH₂)₄CH═CHCH₂CH═CH(CH₂)₇CO(OC₂H₄)_(m)OH,    -   where m=1 to 7;         CH₃(CH₂)₄CH═CHCH₂CH═CH(CH₂)₇CONHCH₂CH₂(OC₂H₄)_(m)OH,    -   where m=1 to 6;        CH₃(CH₂)₄CH═CHCH₂CH═CH(CH₂)₇CO(OC₂H₄)_(m)OCH₂COOH,    -   where m=1 to 6;        CH₃(CH₂)₄CH═CHCH₂CH═CH(CH₂)₇CH₂(OC₂H₄)_(m)OCH₂COOH    -   where m=1 to 6;        CH₃(CH₂CH═CH)₃(CH₂)₇CH₂(OC₂H₄)_(m)OH    -   where m=1 to 7;        CH₃(CH₂CH═CH)₃(CH₂)₇CO(OC₂H₄)_(m)OH,    -   where m=1 to 7;        CH₃(CH₂CH═CH)₃(CH₂)₇CONHCH₂CH₂(OC₂H₄)_(m)OH,    -   where m=1 to 6;        CH₃(CH₂CH═CH₃(CH₂)₇CO(OC₂H₄)_(m)OCH₂COOH,    -   where m=1 to 6;        CH₃(CH₂CH═CH₃(CH₂)₇CH₂(OC₂H₄)_(m)OCH₂COOH,    -   where m=1 to 6.        4.1 Therapeutic Compounds

The invention thus comprehends various compositions for therapeutic (invivo) application, wherein the peptide component of the conjugatedpeptide complex is a physiologically active, or bioactive, peptide. Insuch peptide-containing compositions, the conjugation of the peptidecomponent to the oligomer may be by direct covalent bonding or indirect(through appropriate spacer groups) bonding, and the hydrophilic andlipophilic moieties may also be structurally arranged in the oligomer inany suitable manner involving direct or indirect covalent bonding,relative to one another. A wide variety of peptide species may beaccommodated in the broad practice of the present invention, asnecessary or desirable in a given end use therapeutic application.

While the description is primarily and illustratively directed to theuse of enkephalin as a peptide component in various compositions andformulations of the invention, it will be appreciated that the utilityof the invention is not thus limited, but rather extends to any peptidespecies which is tapable of conjugation to the oligomers hereindescribed, or which is capable of being modified, as for example by theincorporation of a proline residue, so as to enable the peptide to beconjugated to the oligomers described herein.

Accordingly, appropriate peptides include, but or not limited to:adrenocorticotropic hormone, adenosine deaminase ribonuclease, alkalinephosphatase, angiotensin, antibodies, arginase, arginine deaminease,asparaginase, caerulein, calcitonin, chemotrypsin, cholecystokinin,clotting factors, dynorphins, endorphins, endorphins, enkephalins,enkephalins, erythropoietin, gastrin-releasing peptide, glucagon,hemoglobin, hypothalmic releasing factors, interferon, katacalcin,motilin, neuropeptide Y, neurotensin, non-naturally occurring opioids,oxytosin, papain, parathyroid hormone, peptides prolactin, soluble CD-4,somatomedin, somatostatin, somatostatin, somatotropin, superoxidedismutase, thyroid stimulating hormone, tissue plasminogen activator,trypsin, vasopressin, and analogues of such peptides, as well as othersuitable enzymes, hormones, proteins, polypeptides, enzyme-proteinconjugates, antibody-hapten conjugates, viral epitopes, etc.

In another aspect, the therapeutic peptide of the amphiphilicdrug-oligomer conjugates are as described in U.S. Pat. No. 5,641,861,which is incorporated herein by reference, so long as any of suchpeptides contains a lysine residue. Exemplary peptides described thereininclude: Ac-Phe-Arg-Trp-Trp-Tyr-Lys-NH₂ (SEQ ID NO:4);Ac-Arg-Trp-Ile-Gly-Trp-Lys-NH₂ (SEQ ID NO:5);Trp-Trp-Pro-Lys-His-Xaa-NH₂ (SEQ ID NO:6), where Xaa can be any one ofthe twenty naturally occurring amino acids, or Trp-Trp-Pro-Xaa-NH₂ (SEQID NO:7), where Xaa is Lys or Arg; Tyr-Pro-Phe-Gly-Phe-Xaa-NH₂ (SEQ IDNO:8), wherein Xaa can be any one of the twenty naturally occurringamino acids; (D)Ile-(D)Met-(D)Ser-(D)Trp-(D)Trp-Gly_(n)-Xaa-NH₂ (SEQ IDNO:9), wherein Xaa is Gly or the D-form of a naturally-occurring aminoacid and n is 0 or 1, peptides of this formula can be hexapeptides whenGly is absent (n is 0) and heptapeptides when Gly is present (n is 1);(D)Ile-(D)Met-(D)Thr-(D)Trp-Gly-Xaa-NH₂ (SEQ ID NO:10), wherein Xaa isGly or the D-form of a naturally-occurring amino acid; Tyr-A1-B2-C3-NH₂(SEQ ID NO:11), wherein A1 is (D)Nve or (D)Nle, B2 is Gly, Phe, or Trp,and C3 is Trp or Nap; Pm and red{Me_(x)H_(y)N-Tyr-(NMe)_(z)-Tyr-Xaa-NH₂} (SEQ ID NO:12), wherein x and yindependently are 0, 1, or 2 and z is 0 or 1, and wherein Xaa is Phe orD-Phe; Pm and red (Me_(x)H_(y)N-Tyr-(NMe)_(z)-Tyr-Xaa-NHBz1) (SEQ IDNO:53), wherein x and y independently are 0,1, or 2 and z is 0 or 1, andwherein Xaa is Phe or D-Phe; Trp-Trp-Pro-D4-His_(z)-Xaa_(z)-NH₂ (SEQ IDNO:13), wherein z is 0 or 1, D4 is Lys or Arg and Xaa is any one of thenaturally-occurring amino acids.

In still another aspect, the therapeutic peptide of the amphiphilicdrug-oligomer conjugates are as described in U.S. Pat. No. 5,602,099,which is incorporated herein by reference, with the proviso that theconjugation can occur only where there is a free carboxyl or freeN-terminal. Exemplary peptides include: H-Tyr-Tic-Phe-Phe-OH (SEQ IDNO:14); H-Tyr-Tic-Phe-Phe-NH₂ (SEQ ID NO:15); Tyr(NαMe)-Tic-Phe-Phe-OH(SEQ ID NO:16); Tyr(NαCpm)-Tic-Phe-Phe-OH (SEQ ID NO:17);Tyr(NαHex)-Tic-Phe-Phe-OH (SEQ ID NO:18); Tyr(NαEt₂)-Tic-Phe-Phe-OH (SEQID NO:19); H-Dmt-Tic-Phe-Phe-OH (SEQ ID NO:20); H-Dmt-Tic-Phe-Phe-NH₂(SEQ ID NO:21); H-Tyr(3-F)-Tic-Phe-Phe-OH (SEQ ID NO:22);H-Tyr(3-Cl)-Tic-Phe-Phe-OH (SEQ ID NO 23); H-Tyr(3-Br)-Tic-Phe-Phe-OH(SEQ ID NO:24); H-Dmt-TicΨ[CH₂—NH]Phe-Phe-OH (SEQ ID NO:25);H-Dmt-TicΨ[CH₂—NH]Phe-Phe-NH₂ (SEQ ID NO:26);H-Tyr-TicΨ[CH₂—NCH₃]Phe-Phe-OH (SEQ ID NO:27);H-Tyr-TicΨ[CH₂—NH]Hfe-Phe-OH (SEQ ID NO:28);Tyr(NαMe)-TicΨ[CH₂—NH]Hfe-Phe-OH (SEQ ID NO:29); H-Tyr-Tic-Phg-Phe-OH(SEQ ID NO:30); H-Tyr-Tic-Trp-Phe-OH (SEQ ID NO:31);H-Tyr-Tic-Trp-Phe-NH₂ (SEQ ID NO:32); H-Tyr-Tic-His-Phe-OH (SEQ IDNO:33); H-Tyr-Tic-2-Nal-Phe-OH (SEQ ID NO:34); H-Tyr-Tic-Atc-Phe-OH (SEQID NO:35); H-Tyr-Tic-Phe-Phe(pNO₂)—OH (SEQ ID NO:36);H-Tyr-Tic-Trp-Phe(pNO₂)—OH (SEQ ID NO:37); H-Tyr-Tic-Phe-Trp-NH₂ (SEQ IDNO:38); H-Tyr-Tic-Phe-Phe-Val-Val-Gly-NH₂ (SEQ ID NO:39);H-Tyr-Tic-Phe-Phe-Tyr-Pro-Ser-NH₂ (SEQ ID NO:40);H-Tyr-Tic-Trp-Phe-Tyr-Pro-Ser-NH₂ (SEQ ID NO:41); H-Tyr-Tic-Trp-Phe(pNO₂)-Tyr-Pro-Ser-NH₂ (SEQ ID NO:42) andH-Tyr-Tic-Phe-Phe-Leu-Nle-Asp-NH₂ (SEQ ID NO:43).

Abbreviations in the aforementioned peptides of U.S. Pat. No. 5,602,099may be interpreted as follows: Aib=α-aminoisobutyric acid;Atc=2-aminotetralin-2-carboxylic acid; Boc=tert-butoxycarbonyl;Cpm=cyclopropylmethyl; DCC=dicyclohexyl-carbodiimide;D1EA=diisopropylethylamine; Dmt=2,6-dimethyltyrosine; Et=ethyl;Hex=hexyl; Hfe=homophenylalanine; HOBt=1-hydroxybenzotriazole; MVD=mousevas deferens; 1-Nal=3-(1′-naphthyl)alanine;2-Nal=3-(2′-naphthyl)alanine; Phe(pNO₂)=4-nitrophenylalanine;Phg=phenylglycine; Tic=1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid;TIP=H-Tyr-Tic-Phe-OH (SEQ ID NO:44); TIP-NH₂═H-Tyr-Tic-Phe-NH₂ (SEQ IDNO:45); TIPP(Ψ)=H-Tyr-TicΨ[CH₂—NH]Phe-OH (SEQ ID NO:46);TIPP=H-Tyr-Tic-Phe-Phe-OH (SEQ ID NO:14); TIPP-NH₂═H-Tyr-Tic-Phe-Phe-NH₂(SEQ ID NO:15); TIPP(Ψ)=H-Tyr-TicΨ[CH₂—NH]Phe-Phe-OH (SEQ ID NO:47);Tyr(3-Br)=3-bromotyrosine; Tyr(3-Cl)=3-chlorotyrosine;Tyr(3-F)=3-fluorotyrosine; and Tyr(NαMe)═Nα-methyltyrosine.

In another aspect, the peptides are as described in U.S. Pat. No.5,545,719, which is incorporated herein by reference.

Other exemplary peptides include, for example, ACTH-related peptides forinducing neural regeneration, cyclosporin for treating infection,enkephalin analogs for treating pain and drug addiction, MIF-1 fortreating depression, neurotensin for relieving pain, and peptide T fortreating AIDS-associated dementia. Adrenocorticotropic hormone (ACTH)and its analogue peptides are also known to restore the avoidanceleaming caused by removal of the pituitary gland and can also be used totreat passive avoidance conditions.

Particularly preferred peptides are the endogenous and synthetic Opioidpeptides such as the enkephalins. A particlarly preferred Opioid is[Met⁵]Enkephalin (Tyr-Gly-Gly-Phe-Met) SEQ ID NO: 48.

Peptides according to the present invention may be synthesized accordingto any method of sysnthesis known in the art. Such methods include, butare not limited to chemical synthesis techniques and recombinant DNAexpression techniques.

The therapeutic compounds of the present invention can be modified inorder to facilitate coupling to the amphiphilic oligomer. A functionalgroup may be added to the C-terminus or the N-terminus of the peptide orto a side chain of the peptide in order to provide a point of attachmentfor the oligomer.

Alternatively, specific amino acids may be inserted within the aminoacid chain of the peptide therapeutic, or may replace an amino acid ofthe therapeutic or may be added to the C-terminus or N-terminus of thepeptide in order to facilitate attachment of the oligomer where suchmodification does not eliminate the activity of the peptide. Forexample, a proline or alanine residue can be added to the N-terminus ofa therapeutic peptide, such as an enkephalin, such as [met⁵]enkephalin,in order to facilitate attachment of the amphiphilic oligomer.

One skilled in the art would know that one or more amino acids withinthe exemplified peptides could be modified or substituted, as forexample, by a conservative amino acid substitution of one or more of thespecific amino acids shown in the exemplified peptides. A conservativeamino acid substitution change can include, for example, thesubstitution of one acidic amino acid for another acidic amino acid, ofone hydrophobic amino acid for another hydrophobic amino acid or otherconservative substitutions known in the art, including the use ofnon-naturally occurring amino acids, such as Nle for Leu or omithine(Om) or homoArginine (homoArg) for Arg.

In addition to the above types of modifications or substitutions, amimic of one or more amino acids, otherwise known as a peptide mimeticpeptidomimetic, can also be used. As used herein, the term “mimic” meansan amino acid or an amino acid analog that has the same or similarfunctional characteristic of an amino acid. Thus, for example, a(D)arginine analog can be a mimic of (D)arginine if the analog containsa side chain having a positive charge at physiological pH, as ischaracteristic of the guinidinium side chain reactive group of arginine.A peptide mimetic or peptidomimetic is an organic molecule that retainssimilar peptide chain pharmacophore groups as are present in thecorresponding peptide.

The substitution of amino acids by non-naturally occurring amino acidsand peptidomimetics as described above can enhance the overall activityor properties of an individual peptide based on the modifications to theside chain functionalities. For example, these types of alterations canbe employed along with the amphiphilic oligomers of the presentinvention to further enhance the peptide's stability to enzymaticbreakdown and increase the peptide biological activity.

One skilled in the art can easily synthesize the peptides for use astherapeutics in this invention. Standard procedures for preparingsynthetic peptides are well known in the art. The peptides can besynthesized using the solid phase peptide synthesis (SPPS) method ofMerrifield (J. Am. Chem. Soc., 85:2149 (1964), which is incorporatedherein by reference) or using standard solution methods well known inthe art (see, for example, Bodanzsky, M., Principles of PeptideSynthesis 2nd revised ed. (Springer-Verlag, 1988 and 1993), which isincorporated herein by reference). Alternatively, simultaneous multiplepeptide synthesis (SMPS) techniques well known in the art can be used.Peptides prepared by the method of Merrifield can be synthesized usingan automated peptide synthesizer such as the Applied Biosystems 431 A-01Peptide Synthesizer (Mountain View, Calif.) or using the manual peptidesynthesis technique described by Houghten, Proc. Nat. Acad. Sci., USA82:5131 (1985), which is incorporated herein by reference.

Peptides can be synthesized using amino acids or amino acid analogs, theactive groups of which are protected as necessary using, for example, at-butyldicarbonate (t-BOC) group or a fluorenylmethoxy carbonyl (FMOC)group. Amino acids and amino acid analogs can be purchased commercially(Sigma Chemical Co.; Advanced Chemtec) or synthesized using methodsknown in the art. Peptides synthesized using the solid phase method canbe attached to resins including 4-methylbenzhydrylamine (MBHA),4-(oxymethyl)-phenylacetamidomethyl and4-(hydroxymethyl)phenoxymethyl-copoly(styrene-1% divinylbenzene) (Wangresin), all of which are commercially available, or top-nitrobenzophenone oxime polymer (oxime resin), which can besynthesized as described by De Grado and Kaiser, J. Org. Chem. 47:3258(1982), which is incorporated herein by reference.

A newly synthesized peptide can be purified using a method such asreverse phase high performance liquid chromatography (RP-HPLC) or othermethods of separation based on the size or charge of the peptide.Furthermore, the purified peptide can be characterized using these andother well known methods such as amino acid analysis and massspectrometry.

4.2 Synthesis

A general synthesis scheme for the oligomers of the present invention isprovided in FIG. 9, and a general synthesis scheme for attaching sucholigomer to the therapeutic peptides of the instant invention isprovided in FIG. 10.

Several methods of modifying fatty acid to achieve the desired oligomerwill be discussed in further detail with structural illustrations.

In the synthesis of oligomers containing fatty acids and polyethyleneglycols, where the ethylene glycol is connected to the fatty acid in ahydrolysable ester bond, it is desirable to start with the acid chlorideof the fatty acid or its acid anhydride. A desired polyethylene glycolhaving two free hydroxyls at the termini is then treated in inertsolvent with equal molar equivalent of acid chloride or acid anhydride.The glycol unit is first dissolved in inert solvent and treated withorganic base before the addition of the acid chloride or acid anhydride.The product is extracted from the reaction medium and further purifiedusing column chromatograph:

In some instances it is desired to create oligomers that have strongerhydrolysable bond such as amide. The acid chloride or the acid anhydrideof the selected fatty acid is treated with amino derivative ofpolyethylene glycol in a controlled reaction condition to effect onlythe amino residue and not the hydroxyl portion. Other conditions thatensure selectivity is by converting the fatty acid intoN-hydroxysuccinimide ester and reacting with the amino residue of thepolyethylene glycol.

Coupling of the oligomer to the peptide drug is effected by convertingthe free hydroxyl moiety of the oligomer to N-hydroxysuccinimide ester(NSU). N-hydroxysuccinimide group reacts readily with the nucleophilicamino residue of the peptide.

In the synthesis of oligomers in which the lipophilic portion of theoligmers is connected to the hydrophilic portion by ether linkage, thedesired polyethylene glycol (hydrophile) is first protected. One of thetwo free hydroxyis at the termini is protected with a trityl group inpyridine using one mole of trityl chloride. The protected polyethyleneglycol is dissolved in a suitable inert solvent and treated with sodiumhydride. Bromo or tosylate derivative of the lipophilic portion isdissolved in inert solvent and added to the solution of the protectedpolyethylene glycol. The product is treated with a solution ofpara-toluenesulfonic acid in anhydrous inert solvent at roomtemperature. The desired product is extracted in inert solvent andpurified by column chromatography. The structures of the transformationare depicted below:

The lipophilic portion can be alkyl, cholesteryl, adamantyl moieties.

In the synthesis of oligomers where the lipophilic portion of theoligomer is connected to the hydrophilic portion in ether bond and theterminal ends in carboxylic acid moiety, it is desirable to protect thecarboxylic group. Polyethylene glycol having free hydroxyl group at oneend and carboxylic group at the other end is selected. The carboxylicgroup is protected by esterification. The protected polyethylene glycolis dissolved in a suitable inert solvent and treated with sodiumhydride. Bromo or tosylate derivatives of the lipophilic portion isdissolved in inert solvent and added to the solution of the protectedpolyethylene glycol. The product is treated with solution of sodiumhydroxide to liberate free acid. The desired product is extracted ininert solvent and purified by column chromatography. The structures ofthe transformation are depicted below.

The lipophilic portion can be alkyl, cholesteryl or adamantyl moieties.

This group of acidic oligomers can be coupled to peptide drugs by firstreacting the carboxylic group with N-hydroxysuccinimide (NSU) to fromeasily leavable group. A solution of the activated oligomers in inertsolvent is treated with the desired peptide drug dissolved in a suitablesolvent. Inverse addition may be selected.

Sometimes it is desirable to replace the lipophilic moiety withlipophilic sugars. The sugar moiety is first esterified with desiredfatty acid chloride to obtain selective or partial acylation. Theproduct is treated in inert solvent with diacid chloride of desireddicarboxylic acid derivative of polyethylene glycol.

Reaction is conducted with one molar equivalent of each reacting moiety.This reaction leaves one end of the hydrophile bearing acid chloride,which is further converted to N-hydroxysuccinimide ester. The activatedester is reacted with peptide drug in suitable inert solvent.

Where R=fatty acid, alkyl₁₋₂₆, cholesterol or adamantane.

4.3 Therapeutic Methods

The invention provides methods of treatment and prevention byadministration to a subject of an effective amount of an amphiphilicdrug-oligomer conjugate of the invention.

One embodiment of the invention provides for methods of administering apharmaceutical composition which is comprised of a therapeuticallyeffective amount of an amphiphilic drug-oligomer conjugate according tothe present invention.

Methods of introduction include but are not limited to intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, and oral routes. The conjugates may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local.

In certain circumstances, it may be desirable to introduce thepharmaceutical compositions of the invention directly into the centralnervous system by any suitable route, including intraventricular andintrathecal injection; intraventricular injection may be facilitated byan intraventricular catheter, for example, attached to a reservoir, suchas an Ommaya reservoir.

Pulmonary or nasal administration can also be employed, e.g., by use ofan inhaler or nebulizer, and formulation with an aerosolizing agent.

In another embodiment, the conjugates can be delivered in a controlledrelease system. In one embodiment, a pump may be used (see Langer,supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald etal., Surgety 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574(1989)). In yet another embodiment, a controlled release system can beplaced in proximity of the therapeutic target, i.e., the brain, thusrequiring only a fraction of the systemic dose (see, e.g., Goodson, inMedical Applications of Controlled Release, supra, vol. 2, pp. 115-138(1984)).

Other controlled release systems are discussed in the review by Langer(Science 249:1527-1533 (1990)).

The subject is preferably an animal, including, but not limited to,animals such as cows, pigs, horses, chickens, cats, dogs, etc., and ispreferably a mammal, and most preferably human.

4.4 Pharmaceutical Compositions

Exemplary means of administration include oral, parenteral, rectal,topical, sublingual, mucosal, nasal, opthalmic, subcutaneous,intramuscular, intravenous, transdermal, spinal, intrathecal,intra-articular, intra-arterial, sub-arachnoid, bronchial, lymphatic,and intrauterine administration.

The present invention contemplates the use of pharmaceuticalformulations for human medical use which, comprise the vector structuresof the present invention as therapeutic ingredients. Such pharmaceuticalformulations may include pharmaceutically effective carriers, andoptionally, may include other therapeutic ingredients. The carrier orcarriers must be pharmaceutically acceptable in the sense that they arecompatible with the therapeutic ingredients and are not undulydeleterious to the recipient thereof. The therapeutic ingredient oringredients are provided in an amount necessary to achieve the desiredtherapeutic effect, described below.

In another aspect, a pharmaceutical composition is provided tocomprising (1) a mixture of an enkephalin conjugate according to thepresent invention wherein the enkephalin peptide has proline or alanineadded to its N-terminus and an enkephalin conjugate according to thepresent invention which does not have a proline or alanine added to theN-terminus, and (2) a pharmaceutical carrier.

Various delivery systems are known and can be used to administer aconjugate of the invention, e.g., encapsulation microcapsules

As used herein, the term “pharmaceutically acceptable” means approved bya regulatory agency of the Federal or a state government or listed inthe U.S. Pharmacopeia or other generally recognized pharmacopeia for usein animals, and more particularly in humans.

The term “carrier” refers to a diluent, adjuvant, excipient, or vehiclewith which the conjugate is administered.

Such pharmaceutical carriers can be sterile liquids, such as water andoils, including those of petroleum, animal, vegetable or syntheticorigin, such as peanut oil, soybean oil, mineral oil, sesame oil and thelike. Water is a preferred carrier when the pharmaceutical compositionis administered intravenously. Saline solutions and aqueous dextrose andglycerol solutions can also be employed as liquid carriers, particularlyfor injectable solutions.

Suitable pharmaceutical excipients include starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene glycol, water, ethanol and the like.

The composition, if desired, can also contain minor amounts of wettingor emulsifying agents, or pH buffering agents.

The compositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations andthe like. The composition can be formulated as a suppository, withtraditional binders and carriers such as triglycerides.

Oral formulation can include standard carriers such as pharmaceuticalgrades of mannitol, lactose, starch, magnesium stearate, sodiumsaccharine, cellulose, magnesium carbonate, etc. Examples of suitablepharmaceutical carriers are described in “Remington's PharmaceuticalSciences” by E. W. Martin.

Such compositions will contain a therapeutically effective amount of thedrug-oligomer conjugate, preferably in purified form, together with asuitable amount of carrier so as to provide the form for properadministration to the patient. The formulation should suit the mode ofadministration.

The mode of administration and dosage forms will of course affect thetherapeutic amounts of the compounds which are desirable and efficaciousfor the given treatment application. A therapeutically effective amountis an amount necessary to prevent, delay or reduce the severity of theonset of disease, or an amount necessary to arrest or reduce theseverity of an ongoing disease. It will be readily apparent to one ofskill in the art that this amount will vary based on factors such as theweight and health of the recipient, the type of cells being transformed,the mode of administration of the present compositions and the type ofmedical disorder being treated.

The dosage can be presented in the form of tablets, syrups, losenges,elixirs, suspensions, and/or emulsions.

Accessory ingredients may, without limitation, include diluents,buffers, flavoring agents, disintegrants, surfactants, thickeners,lubricants, preservatives, and/or antioxidants.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings.

Typically, compositions for intravenous administration are solutions insterile isotonic aqueous buffer. Where necessary, the composition mayalso include a solubilizing agent and a local anesthetic such aslignocaine to ease pain at the site of the injection. Generally, theingredients are supplied either separately or mixed together in unitdosage form, for example, as a dry lyophilized powder or water freeconcentrate in a hermetically sealed container such as an ampoule orsachette indicating the quantity of active agent.

Where the composition is to be administered by infusion, it can bedispensed with an infusion bottle containing sterile pharmaceuticalgrade water or saline. Where the composition is administered byinjection, an ampoule of sterile water for injection or saline can beprovided so that the ingredients may be mixed prior to administration.

The conjugates of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed with freeamino groups such as those derived from hydrochloric, phosphoric,acetic, oxalic, tartaric acids, etc., and those formed with freecarboxyl groups such as those derived from sodium, potassium, ammonium,calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

The amount of the Conjugate of the invention which will betherapeutically effective in the treatment of a particular disorder orcondition will depend on the nature of the disorder or condition, andcan be determined by standard clinical techniques. In addition, in vivoand/or in vitro assays may optionally be employed to help identifyoptimal dosage ranges.

For example, suitable doses of a an enkephalin conjugate for analgesiamay genarally be in the range of from 1 mg/kg to 20 mg/kg, preferably 3mg/kg to 15 mg/kg, more preferably 5 mg/kg to 7 mg/kg.

Effective doses may be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems.

Suppositories generally contain active ingredient in the range of 0.5%to 10% by weight; oral formulations preferably contain 10% to 95% activeingredient.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

5. EXAMPLES 5.1 Synthesis

5.1.1 Trirthylene Glycol Monohexadecyl Ester

Palmitic anhydride (5.00 g; 10.104 mmol) was dissolved in dry THF (20mL) and 3 mol excess of dry pyridine and the solution was stirred atroom temperature. To the stirring solution, triethylene glycol (1.5 g;10.104 mmol) was added slowly. After stirring for 1 h, THF was removedunder reduced pressure at room temperature and the reaction mixture waspoured into ice cold 10% sulfuric acid. The aqueous layer was extractedwith ethyl acetate (30 ml×3). Combined organic layer was sequentiallywashed with water, brine and dried over MgSO₄ and filtered. Afterevaporation gave pure product, single spot on TLC.

5.1.2 Succinimidyl Trirthylene Glycol Monohexadecyl Ester:

To a stirring solution of teg-palmitate (1 g; 2.57 mmol),dimethylaminopyridine (0.313 g; 2.57 mmol) in dry THF was addedN,N′-disuccinimidyl carbonate (0.691 g) in one portion. The reactionmixture was stirred overnight at room temperature. The organic solventwas removed under reduced pressure and reaction mixture was diluted withethyl acetate, washed with 1 N hydrochloric acid (10 mL×2), water andbrine. The solvent was dried over MgSO₄ filtered and evaporated to leavewhite solid.

5.1.2.1 Determination of Activity

The succinimidyl reactivity was determined by conjugating it withinsulin and it was found to be 67%.

5.1.3 Succinimidyl Trirthylene Glycol Monohexadecyl Ether

To a cold stirring solution of phosgene (10.0 mL; 20% solution intoluene) under nitrogen, a solution of triethylene glycol monohexadecylether (1.5 g; 4.00 mmol) in dry dichloromethane (4 mL) was added. Thereaction mixture was stirred at 0° C. for 2 h at room temperature.Excess of phosgene was distilled off using water aspirator, passingthrough cold solution of dilute NaOH.

The reaction flask was cooled in ice bath and equimolar quantity oftriethyl amine and a solution of hydroxysuccinimide, dissolved inminimum quantity of THF was added slowly. The reaction mixture wasstirred at room temperature for 12 h. The solvent was removed completelyat 25° C. and residue was redisolved in ethyl acetate, washed withwater, brine, dried over MgSO₄ and evaporated to give pure succinimidylderivative

5.1.3.1 Determination of Activity

The succinimidyl reactivity was determined by conjugating it withinsulin and it was found to be 62.5%.

5.2 Conjugation of Compound 2 & 4 with Met-enkephalin

5.2.1 General Procedure for Conjugation

5.2.1.1 Scheme

5.2.1.2 General Procedure

To a stirring solution of met-enkephalin SEQ ID NO: 48 (0.130 g; 0:1854mmol) in 5 mL of DMF-DCM (2:1) was added TEA (25 μL). The reactionmixture was cooled to 10° C. and a solution of palmityl-teg-nsu orcetyl-teg-nsu dissolved in 1 mL of DCM was added in one portion. Thereaction mixture was stirred for 2 h at 10° C. The solvent was removedunder reduced pressure and the residue was redissolved in dry ethylacetate. After evaporation of the solvent 0.310 g conjugated enkephalinwas obtained. HPLC showed mono & diconjugate in the ratio of 3:1.

5.3 Synthesis of Cetyl-PEG₂; It's Activation & Conjugation withProtected (BOC) Leuenk

To a suspension of NaH (4.00 g: 0.1 mol) in dry THF (300 mL) at 10° C.was added diethylene glycol in one portion. The cooling bath was removedand reaction mixture was stirred at room temperature for 2 h. Al the endthe reaction mixture was cooled to 10° C. and bromohexadecane (29 g)0.095 mol) was added in one portion. The cooling bath was removed andthe reaction was stirred at room temperature for 4 h. The solvent wasremoved under reduced pressure and crude was admixed with water andextracted with ethyl acetate (30 mL×3). The combined organic extract wassequentially washed with water, brine, dried over MgSO₄ and evaporatedto leave white solid powder, single spot on TLC and single molecular ionpeak.

5.3.1 Cetyl-PEG₂-NSU

To a cold stirring solution of phosgene (10.0 mL; 20% solution intoluene) under nitrogen, a solution of cetyl-PEG₂OH (1.3 g; 4.00 mmol)in dry dichloromethane (5 mL) was added. The reaction mixture wasstirred at 0° C. for 1 hr and 2 h at room temperature. Excess ofphosgene was distilled off using water aspirator, passing through coldsolution of dilute NaOH.

The reaction flask was cooled in ice bath and equimolar quantity oftriethyl amine and a solution of hydroxy succinimide, dissolved inminimum quantity of THF was added slowly. The reaction mixture wasstirred at room temperature for 12 h. The solvent was removed completelyat 25° C. and residue was redissolved in ethyl acetate, washed withwater, brine, dried over MgSO₄ and evaporated to give pure succinimidylderivative.

5.3.2 Determination of Activity

The succinimidyl reactivity was determined by conjugating it withinsulin and it was found to be 83.5%.

5.4 Conjugation of Succinimidyl Cetyl-PEG2 with BOC-LEU . . . ENK . . .LYS-OH SEQ ID NO: 51

Boc-Leu . . . enk . . . lys-OH SEQ ID NO: 51 (100 mg; 0.125 mmol) wasdissolved in 5 mL of DMF:DCM(1:1) and stirred at 10° C. under nitrogen.To this clear solution TEA (17.5 μL) and a solution of succinimidylcetyl-PEG₂, dissolved in 1 mL of DCM were added.

After 1.5 h (TLC showed single product) the solvent was removed underreduced pressure at room temperature and reaction mixture was admixedwith water and extracted with ethyl acetate (10 mL×3). The organicextract was sequentially washed with water, brine, dried and evaporatedto a solid.

5.4.1 Purification of Derivatized BOC-LEU . . . ENK . . . LYS-OH SEQ IDNO: 51 ON Silica Gel Column

The derivatized blocked enkephalin was purified on silica gel columnusing methanol-chloroform (5% methanol-chloroform) mixture as an elutingsolvent. After evaporation of desired fraction 100 mg pure compound wasobtained. A product yield of 100 mg was obtained after removal of thesolvent.

5.4.2 Deblocking of Butyloxycarbonyl Group from Derivatized LEU . . .ENK SEQ ID NO: 52

Derivatized Boc-Leu . . . enk SEQ ID NO: 52 (100 mg: 0.0866 mmol) wastreated with 0.4 ml of TFA-DCM (1:1) for 30 min. at room temperature.The solvent was removed under reduced pressure. The solid wasredissolved in 2 ml of methanol, filtered and evaporated; 80 mg of pureproduct was obtained.

5.5 Synthesis of Amphiphilic Oligomer-Enkephalin Conjugates

5.5.1 A General Scheme for Synthesis of Non-hydrolyzable andHydrolyzable Conjugates

One-hundred milligrams of enkephalin (100 mg; 0.142 mmol) was dissolvedin dry dimethylformamide (5 mL) at room temperature. P-nitrophenol orN-hydroxysuccinimide activated (carbonate or ester) of amphiphilicoligomer (1.1 mole equivalent) was dissolved in 1 mL tetrahydrofuran andadded to above solution and stirred at room temperature over 1.5 hours.The extent of the reaction was monitored by a reverse phase (C-18) HPLCusing isopropanol/water (4% trifluoroacetic acid) gradient system.Reaction mixture was evaporated under reduced pressure and the contentswere dissolved in an isopropanol-water mixture. This mixture waspurified on a 22 mm preparative HPLC column (C-8) with a solventgradient system made of either isopropanol/water (0.1% trifluoroaceticacid) or acetonitrile/water (0.1% trifluoroacetic acid to give puremonoconjugated and diconjugated enkephalins. The solvent was evaporatedat low temperature (<20° C.) to give dry produce. The purity of theproduct was analyzed by reverse phase analytical HPLC, and the MWinformation was obtained by MALDI (TOF)-mass spectral technique.

5.5.2 Synthesis of Cholesterol-PEG₂ Hydrolyzable Amphiphilic Oligomer

PEG₂ diacid (3,6,9-trioxaundecanoic diacid, 10 g) was dissolved in drychloroform (50 mL) and added dropwise to oxalychloride at roomtemperature under dry condition in the presence of catalytic amount ofdimethylformamide. The reaction was stirred or 6 hours and the solventand excess of reagent was stripped off to give an oily residue. Aboveresidue was dissolved in chloroform (50 mL) and to this was addedcholesterol (1.05 mole equivalent) in chloroform (50 mL) andtriethylamine (1 mole equivalent) over 30 minutes at 5° C. The reactionwas stirred at 15° C. over 2 hours. To this was addedN-hydroxysuccinimide (1 mole equivalent) in chloroform (50 mL) andfollowed by triethylamine (1 equivalent) at 5° C. and allowed to stirovernight. Solvent was stripped off and the product was extracted withethylacetate. Crude product was purified on a silica gel column with1:10 methanol/chloroform solvent system to obtain activated amphiphilicoligomer in 80% yield.

5.6 Molecular Weight Information of Enkephalin Conjugates Obtained byMaldi (TOF)-MS

Enkephalin Conjugate Expected M.W. Observed M.W. Cholesterol-PEG₂ 12741275 DHA-PEG₂ 1144.4 1144.3 Linolenic-PEG₂ 1093.4 1093.3 Cetyl-PEG₂ Avg.1059 Avg. 1032 Palmitate-PEG₃ 1116 1115.6 Cetyl-PEG₃ 1101 1101.12

These results demonstrate that the reactions resulted in monoconjugates,i.e., each peptide was coupled to only one oligomer. It is significantto note that a single conjugate is sufficient to impart amphiphilicproperties.

5.7 Stability of Met Enkephalin-LYS SEQ ID NO: 49 (Enkephalin) and ItsAmphiphilic Oligomer Conjugates in Rat Brain Homogenate

Met enkephalin-lys SEQ ID NO: 49 and its conjugates (Cetyl-PEG₂,Cetyl-PEG₃ and Palmitate-PEG₃) were incubated in 2% rat brainhomogenate. Samples were drawn over time intervals and the amount of thesubstance remaining was measured by a HPLC method. Followingexperimental procedure was used for the study.

Procedure: A 2% rat brain homogenate was prepared by homogenizingfreshly perfused (PBS buffer) rat brair in PBS buffer (pH 7.4). Two 3-mLaliquots of the homogenate were equilibrated at 37° C. in a water bath.To one unmodified enkephalin was added and to other modified (conjugate)was added, resulting in a final concentration of 60 μg/ml of peptide. Attime 0, 1, 2, 3, 5,15, 30, and 60 minutes, 200 μL of aliquot waswithdrawn and quenched with 200 uL of the quenching agent (1%trifluoroacetic acid in acetonitrile/isopropanol or 1% trichloroaceticacid in water). The sample solutions were vortexed and centrifuged at7000 RPM. The supernatant was analyzed by a HPLC method using a gradientof 10 to 100% isopropanol/water (0.1% trifluoroacetic acid) on a C-18column.

FIG. 2 shows the stability of the cetyl-PEG₂-enkephalin SEQ ID NO: 1conjugate as compared to free met-enkephalin-lys SEQ ID NO: 49. FIG. 3shows the stability of the cetyl-PEG₃-enkephalin SEQ ID NO: 1 ascompared to met-enkephalin-lysine SEQ ID NO: 49. FIG. 4 showspalmitate-PEG₃-enk SEQ ID NO: 1 (hydrolyzable) conjugate as compared tomet-enkephalin-enk SEQ ID NO: 49.

5.7.1 Extraction and Detection of Enkephalin Conjugates from the Brainof Dosed Rats

5.7.1.1 Procedure

The following procedure was used to identify the presence of conjugatefrom the brain specimen of animals dosed with 5 mg/kgcetyl-PEG₂-enkephalin SEQ ID NO: 1.

After 10 minutes of dosing, the brain of the animal was perfused with1.5% triflouroacetic acid in PBS solution, and the brain was removed andfrosen at −70° C. The brain was homogenized with 1 mL of 1.5%triflouroacetic acid in PBS solution and the homogenate was extractedwith acetonitrile/isopropanol solution. The extract was treated withsaturated sodium chloride solution and frozen at −20° C. for 2 hours.The organic layer was isolated and centrifuged at 4000 RPM. Thesupernatant was evaporated and the resulting residue was reconstitutedin acetonitrile/isopropanol/water mixture. The reconstituted solutionwas analyzed by HPLC using a gradient of 10 to 100% isopropanol/water(0.1% trifloiroacetic acid) on a C-18 column. The presence and theconcentration of cetyl-PEG₂-enkephalin SEQ ID NO: 1 conjugate in theextract were measured by comparing the retention time and the peak areaof standard solution under the same analytical condition. The resultsare presented in FIGS. 5A to 5D.

5.7.1.2 Results

The results demonstrate that monoconjugates were isolated from braintissue. FIG. 5A shows a peak produced by cetyl enkephalin SEQ ID NO: 1standard, while 5B shows a corresponding peak demonstrating that cetylenkephalin SEQ ID NO: 1 was actually present in the brain exact. Incontrast, neither the vehicle (FIG. 5C) nor the unconjugated enkephalinSEQ ID NO: 49 (FIG. 5D) showed a corresponding peak.

5.8 Rat Paw-hot Plate Test

5.8.1 Animals

Adult, male Sprague-Dawley rats weighing 150-175 g were obtained fromCharles River Breeding Laboratories (Raleigh, NC) and used for allanimal studies. Rats were housed in hanging wire-bottomed cages in avivarium equipped with a 12:12 light:dark cycle and humidity wasmaintained between 45-65% with a room temperature of 72±2° C. Rats wereprovided Purina Rodent Chow and tap water ad libitum.

5.8.2 Methods

Met-enkephalin-lys SEQ ID NO: 49 and met-enkephalin-lys SEQ ID NO: 1derivatives were assessed for analgesic activity by rat paw-hot plateassay. Rats were given an injection of naloxone at 0.5 mg/kg (s.c.) thenadministered a single administration of cetyl-enkephalin SEQ ID NO: 1 bythe tail vein 10 minutes later at a dose of 5.0 mg/kg. The results asgraphically displayed in FIG. 6 demonstrate that Naloxone, an μ-receptoranatagoist prevents competitively inhibits binding ofcetyl-PEG₂-enkephalin SEQ ID NO: 1, thus demonstrating that at leastpart of the activity of cetyl-PEG₂enkephalin SEQ ID NO: 1 is attributeto binding at the Opioid μ-receptor.

In a separate study, rats were administered cetyl-enkephalin SEQ ID NO:1 (5.0 mg/kg, i.v.) or clonidine (0.125 mg/kg, i.v.).

The latency to rat paw withdrawal from the hot plate was measured by aHot Plate Analgesia Meter (Harvard Apparatus Ltd., Kent, England). Thetemperature of the hot plate was set and calibrated at 52° C. and ratswere removed from the heat stimulus by 36 seconds after placement.Latency trials were terminated when the animal was either licking a hindpaw or initiating a jump from the plate. Baseline measurements werecollected 1 hour prior to drug administration and at various timespost-injection, dependent upon the study conducted. All hot platetesting was terminated by 1 hour after drug dosing.

5.8.3 Results

The results are displayed in the following tables and in the Graph ofFIG. 6. The results demonstrate that while 20 mg/kg enkephalin SEQ IDNO: 47 alone has 0% analgesic effect as compared to morphine as abaseline, the enkephalin conjugates of the present invention had stronganalgesic effects and one conjugate, DHA-PEG-ENK had 130% of theanalgesic effect of morphine. The graph of FIG. 7 shows thatCETYL-PEG-ENK produces a response and duration comparable to that ofclonidine, an α-adrenergic receptor agonist.

ANALGESIC EFFECT OF ENKEPHALIN CONJUGATES IN RATS Mean Analgesia asCompared with Dose Number Morphine at 3 mg/Kg* Drug or Conjugate (mg/kg)of Rats @ 5 min @ 30 min Morphine 3 8 100% 100% Enkephalin SEQ ID no:4920 7 0% 0% Cetyl-PEG-ENK SEQ ID no:1 5 8 84% 75% DHA-PEG-ENK SEQ ID no:120 8 130% 67% Cholesterol-PEG- 5 8 80% 68% ENK SEQ ID no:1Linolenic-PEG-ENK 10 8 77% 73% SEQ ID no:1

5.9 Agonist-Stimulated [³⁵S] GTPγS Binding in Brain Sections

5.9.1 Materials

Male Sprague-Dawley rats (200 g) were purchased from Zivic-Miller(Zelienople, Pa.). [³⁵S]GTPγS (1250 Ci/mmol) was purchased from NewEngland Nuclear Corp. (Boston, Mass.). [D-Ala²,N-Me-Phe⁴,Gly⁵-ol]-enkephalin (DAMGO), adenosine deaminase, and GDP wereobtained from Sigma Chemical Co. (St. Louis, Mo.). Reflections®autoradiography film was purchased from New England Nuclear Corp.(Boston, Mass.). All other reagent grade chemicals were obtained fromSigma Chemical Co. or Fisher.

5.9.2 Agonist-Stimulated [³⁵S] GTPγS Binding in Brain Sections.

Agonist-stimulated [³⁵S]GTPγS autoradiography was performed as describedby Sim et al. Proc. Nat'l. Acad. Sci. USA 1992 Pg. 7242-7246. Animalswere sacrified by decapitation and brains were removed and frozen inisopentane at 50° C. Coronal and horizontal brain sections were cut on acryostat maintained at 20° C. Sections were incubated in assay buffer(50 mM Tris-HCl, 3 mM MgCl₂, 0.2 mM EGTA, 100 mM NaCl, pH 7.4) at 25° C.for 10 min. Sections were then incubated in assay buffer containing 2 mMGDP, protease inhibitor cocktail (10 μl/ml of a solution containing 0.2mg/ml each of bestatin, leupeptin, pepstatin A and aprotinin), andadenosine deaminase (9.5 mU/ml) at 25° C. for 15 min. Sections were thenincubated in assay buffer with GDP, 0.04 nM [³⁵S]GTPγS and appropriateagonist at 25° C. for 2 hours. The agonist were 10 μM DAMGO, 10 μMcetyl-enkephalin SEQ ID NO: 1 and 10 μM cetyl-TEG-enkephalin SEQ IDNO: 1. Based binding was assessed in the absence of agonist. Slides wererinsed twice for 2 min each in cold Tris buffer (50 mM Tris-HCl, pH 7.4)and once in deionized H₂O. Slides were dried overnight and exposed tofilm for 72 hours. Films were digitized with a Sony XC-77 video cameraand analyzed using the NIH IMAGE program for Macintosh computers.

5.9.3 Results

Results show that cetyl-TEG-enkephalin SEQ ID NO: 1 stimulates of[³⁵S]GTPγS binding. The anatomical distribution of the binding isconsistent with that of μ Opioid receptors. These results demonstratethat cetyl-TEG-enkephalin SEQ ID NO: 1 does not simply bind the receptorbut also activates the receptor, causing the receptor to bind toG-protein. This activation provides further corroborative evidence thatcetyl-TEG-enkephalin SEQ ID NO: 1 directly stimulates analgesia.

1. A method for inducing analgesia in a subject, the method comprisingdelivering across the blood brain barrier of the subject, into thesubject's central nervous system, a therapeutically effective amount ofan amphiphilic drug-oligomer conjugate having a formula:


2. The method of claim 1 wherein the subject is a human.
 3. The methodof claim 1 wherein the amphiphilic drug-oligomer conjugate isadministered to the subject orally.
 4. The method of claim 1 wherein theamphiphilic drug-oligomer conjugate is administered to the subjectintravenously.
 5. The method of claim 1 wherein the amphiphilicdrug-oligomer conjugate is administered to the subject by a routeselected from the group consisting of pulmonary, intradermal,intramuscular, subcutaneous, and intranasal.
 6. The method of claim 1wherein the amphiphilic drug-oligomer conjugate is administered to thesubject as a component of a pharmaceutical composition.
 7. The method ofclaim 1 wherein the amphiphilic drug-oligomer conjugate is administeredto the subject as a component of a pharmaceutical composition formulatedfor oral administration.
 8. The method of claim 1 wherein theamphiphilic drug-oligomer conjugate is administered to the subject as acomponent of a pharmaceutical composition formulated for intravenousadministration.
 9. The method of claim 1 wherein the amphiphilicdrug-oligomer conjugate is administered to the subject as a component ofa pharmaceutical composition formulated for administration by a routeselected from the group consisting of pulmonary, intradermal,intramuscular, subcutaneous, and intranasal.